Two features characterize the pathogenesis of secondary hyperparathyroidism (HPT). The first is an inadequate control of secretion of parathyroid hormone (PTH) from the parathyroid cells, and the second is an increased rate of parathyroid cell proliferation. While the regulation of secretion has been extensively studied1, parathyroid proliferation and the factors influencing this process are less well understood. Calcium and active vitamin D and their receptors are involved in both processes.
Calcium regulates PTH secretion via receptors in the cell membrane. The best established calcium receptor is the seven-transmembrane receptor, CaR2,3,4,5. Another possible calcium-sensing receptor is the large protein gp330 or CAS, a member of the low density lipoprotein (LDL)-receptor superfamily6,7,8,9,10. Studies on the secretion of PTH from parathyroid cells of secondary HPT in vitro have demonstrated a shift in set point, where the PTH secretion is inappropriately high in relationship to the extracellular calcium level11. One reason for a shift in set point could be inactivating mutations in CaR. However, such mutations have only been demonstrated in familial hypocalciuric hypercalcemia, and not in primary or secondary HPT4. An alternative explanation for the shift in set point would be a decreased number of receptors in the parathyroid cells. Previous studies have shown reduced expression of CaR mRNA and protein both in adenomas from patients with primary HPT and in hyperplastic parathyroid glands from patients with secondary HPT12,13,14. A reduced expression of CAS has been demonstrated in the enlarged glands of secondary HPT15. The reduction in CAS has been shown to parallel the increase in set point for PTH secretion, and antibodies directed against CAS block the response to extracellular calcium in vitro7. Although many findings have suggested a role for CAS in the regulation of PTH secretion, definitive proof has been difficult to obtain since functional expression studies in, for example, Xenopus laevis oocytes, have been impossible to perform because of the very large size of the CAS mRNA.
Calcium has an effect not only on PTH secretion, but also on transcription of the PTH gene mediated via calcium response elements in the upstream flanking region of the PTH gene16,17,18.
The role of CaR and CAS in parathyroid cell proliferation is less well understood. However, calcium also seems to have a direct effect on proliferation, and this effect is probably mediated via calcium receptors on the cell surface.
The relative importance of calcium in relation to vitamin D is difficult to determine. Vitamin D inhibits transcription of the PTH gene via an intranuclear receptor protein, the vitamin D receptor (VDR)19,20. In this manner, vitamin D also influences PTH secretion. Vitamin D inhibits parathyroid cell proliferation21, whereas the combination of low calcium and low vitamin D stimulates parathyroid cell proliferation22. Several studies have demonstrated reduced levels of VDR in hyperplastic parathyroid glands of secondary HPT23,24,25. The most prominent decrease was seen in the largest glands and particularly in nodular hyperplasia26.
Intermittent intravenous 1,25(OH)2D3 therapy is traditionally used in patients with secondary HPT to suppress PTH secretion27 and diminish hyperplasia28. For unknown reasons, some patients develop unresponsiveness to this treatment. This resistance is thought to be caused at least in part by the reduced VDR density of the parathyroid glands.
It is hypothesized that parathyroid cells initially proliferate polyclonally. Diffuse hyperplasia is then followed by monoclonal proliferation of certain cells within the diffuse hyperplasia resulting in nodular hyperplasia29. Nodular hyperplasia is considered to be present in more severe secondary hyperparathyroidism. In line with this is the fact that transplantation of nodular parathyroid tissue to the forearm as part of surgical treatment of secondary HPT results in fewer good results than transplantation of diffusely hyperplastic parathyroid tissue30.
Until the recent publication by Yano et al on the association between decreased CaR expression and proliferation of parathyroid cells in secondary HPT31, there has been no report analyzing the relationship between CaR and VDR in the same specimens. We have studied the mRNA expression of CaR, CAS, VDR, and PTH in a large number of hyperplastic glands from patients with secondary HPT, and demonstrate a highly variable expression of these mRNAs that is apparently independent of each other. It seems as if each clone of parathyroid cells has its own expression pattern, a finding that may explain the variable clinical course of secondary HPT.
METHODS
Patient material
The study includes a total of 36 hyperplastic parathyroid glands from 18 patients operated on for secondary HPT at the Karolinska Hospital Tables 1 and 2. Fifteen patients had advanced chronic renal failure. One was admitted with acute renal failure. One had a renal failure of not-yet-diagnosed reason, and one had hypophosphatemic rickets with normal serum creatinine. At the time of their operation, 11 patients were receiving hemodialysis. Two were receiving peritoneal dialysis, and two had functioning renal transplants. All patients were hypercalcemic with a median preoperative albumin corrected serum calcium concentration of 2.83 mmol/L (range 2.58 to 3.11) and a median preoperative serum intact PTH of 288 ng/L (range 83 to 1860). Median serum phosphate was 1.55 mmol/L (range 0.60 to 2.90). Median total weight of the parathyroids removed in each patient was 2078 mg. The median weight of the individual glands analyzed with in situ hybridization was 648 mg (range 95 to 3077 mg). The histopathological investigation showed diffuse hyperplasia of all glands in one patient, nodular hyperplasia in seven patients, and a mixed pattern of diffuse as well as nodular hyperplasia in ten patients. A total of 29 glands showing nodular hyperplasia and seven glands showing diffuse hyperplasia were analyzed.
Table 1 - Clinical characteristics of the 18 patients with secondary hyperparathyroidism in the study.
Full table Table 2 - Weight and histopathological diagnosis of the hyperplastic glands from the 18 patients in the study.
Full table Fifteen patients had received treatment with vitamin D, either 1
(OH)D3 or 1,25(OH)2D3. In nine cases, the treatment was intermittent intravenous and in four cases oral. Two patients had received both intravenous and oral treatment during the course of the disease. In most cases, this treatment was not given during the last three weeks before the operation due to hypercalcemia. However, in four cases, treatment was stopped only a few days before the surgery Table 1.
Biopsies of normal parathyroid tissue from three patients operated on for sporadic primary hyperparathyroidism were analyzed for comparison.
The tumors and biopsies were snap frozen in liquid nitrogen after removal and stored at -70°C until analysis. The study was approved by the local ethics committee of the Karolinska Hospital.
mRNA in situ hybridization
The cellular expression of mRNA was studied using in situ hybridization. Oligonucleotide probes with sequences complementary to mRNAs encoding for VDR (nt 1257–1296; GenBank accession number J03258.1), CaR (nt 1371–1410, GenBank/EMBL Data Bank accession number U20759 and U20760), CAS (nt 666–705, GenBank/ EMBL Data Bank accession number S73145) and for PTH (nt 402–432, GenBank accession number J00301.1) were synthesized. To verify the presence of mRNA an oligonucleotide complementary to GAPDH (nt 1149–1193; GenBank/EMBL Data Bank accession number M33197), mRNA was also synthesized. Also, a sense probe (corresponding to the inverted sequence between nt 2504 and 2545 of IgF2; EMBL Data Bank accession number X03562.1) was included as a negative control. The oligonucleotides were labeled at the 3' end with [35S]deoxyATP (NEN, Life Science Products, Boston, MA, USA) using terminal deoxynucleotidyl transferase (Amersham Pharmacia Biotech, Piscataway, NJ, USA) followed by a purification step using the QIAquick nucleotide removal kit (Qiagen, Hilden, Germany).
Consecutive cryostat sections of hyperplastic parathyroids and normal biopsies were cut and thaw-mounted onto SuperFrostPlus (Menzel-Gläser, Hannover, Germany) slides. Hybridization was then performed essentially as described by Dagerlind et al32. The hybridization solution containing the labeled probe was spread out on the sections, and the slides were then covered with parafilm, placed in a humified box, and incubated overnight at 42°C. After hybridization, the sections were sequentially rinsed in five changes of 1 
SSC at 60°C for 60 minutes, put on the bench for one hour to cool, and then rinsed in distilled water and dehydrated in 70, 95, and 99% ethanol. After air drying, the sections were exposed to Hyperfilm beta-max x-ray film (Amersham, CEA AB, Uppsala, Sweden) for seven days. Eight standards (Amersham International, Buckingshamshire, UK) to construct a gray level as a function of the activity concentration were included. The x-ray films were then developed, and the semiquantification of film autoradiograms was carried out. Gray levels corresponding to the eight standards were determined, and the analysis was made using the NIH Image program (developed at the U.S. National Institutes of Health and available on the internet at http://rsb.info.nih.gov/nih-image/). Care was taken to get the optimal gray scale and to exclude fat cells, connective tissue, and vessels when measuring. Only homogenous areas consisting of parathyroid parenchymal cells were measured. This procedure has been shown to be reasonably reproducible with a variation coefficient of 14%13. In nodular parathyroids, nodules and internodular areas were measured separately Table 2. Only nodules that could be identified in most analyzed sections (that is, mainly large nodules) were selected for measurement.
Statistical analyses
Statistical analysis was performed using the StatView 4.02 software. An analysis of variance table was used to evaluate correlations between variables. Comparisons between nodules and internodular areas from the same glands were made using the Wilcoxon signed-rank test. P values <0.05 were considered significant.
RESULTS
Thirty-six hyperplastic parathyroid glands from 18 patients with hypercalcemic secondary HPT were analyzed with in situ hybridization for expression of GAPDH, CaR, CAS, VDR, and PTH mRNAs. The clinical information for each case and gland is detailed in Tables 1 and 2.
Overview of expression levels
No signal was detected with the sense probe in any tissue analyzed, supporting the specificity of the results obtained with the antisense probes. Since all glands investigated were positive for GAPDH, no specimen was excluded from analysis due to suspicion of increased degradation of mRNA due to RNAse activity.
A great variability was seen in the expression of the different mRNAs, not only between, but also within the analyzed hyperplastic glands Figure 1a. The mean levels of GAPDH, CaR, CAS, VDR, and PTH mRNA in all measured areas were 170 
81 (N = 42), 290 225 (N = 38), 32 26 (N = 31), 121 70 (N = 42), and 145 106 (N = 42) nCi/g (mean SD), respectively. When compared with the few available biopsies of normal parathyroid glands (N = 2 to 3) run in the same experiment, the levels of GAPDH and CaR mRNA were in the same order of magnitude, whereas the levels of CAS, VDR, and PTH mRNA were lower in the hyperplastic glands (median 23, 41, and 35%, respectively; Figure 2). No statistical evaluation of the relationship between hyperplastic and normal glands was made due to the low number of controls.
Figure 1.
(A) Autoradiogram of consecutive sections of three glands from patient 1 and normal biopsies (upper row only). Frozen sections were hybridized with oligonucleotide probes complementary to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calcium receptor (CaR), calcium-sensing receptor (CAS), vitamin D receptor (VDR), and parathyroid hormone (PTH) mRNA, and then exposed to x-ray film. Nodular areas are denoted N1 to N6 and internodular areas I1 to I3. A highly variable expression of all mRNAs is shown.
(B and C) Autoradiogram of consecutive sections hybridized with probes complementary to GAPDH, CaR, CAS, VDR, and PTH mRNA showing three glands where the expression of VDR mRNA is high and of PTH mRNA low (B) and vice versa (C). In patient 14, a nodule shows high expression of VDR mRNA (B).
Full figure and legend (98K)Figure 2.
Box plot of GAPDH, CaR, VDR and PTH mRNA expression in 36 parathyroid tumors from patients with secondary hyperparathyroidism in relationship to the percent of normal biopsies. The box represents the 25th to the 75th percentile, and the whiskers represent the 10th and the 90th percentiles. The circles represent single values outside the 10th and 90th percentiles.
Full figure and legend (26K)When the expression of GAPDH mRNA was compared with the expression of the other mRNAs, measured in the same defined area of consecutive sections, no correlation was found. A positive correlation was found between the expression of CaR and CAS mRNA (P = 0.02; Figure 3). Otherwise, no correlations between CaR, CAS, VDR, and PTH mRNAs were found. When all measured areas in the nodular glands were compared with glands exhibiting diffuse hyperplasia, no difference was seen with regard to any of the analyzed mRNAs. There was a positive correlation between tumor weight and the "average expression" of CaR mRNA in nodular hyperplasia (P = 0.01). "Average expression" was estimated by calculation of the mean of the expression in measured areas in a single gland, irrespective of the size of those areas. Otherwise, no correlations between tumor weight and the expression of different mRNAs were found.
Figure 3.
Scattergram showing the correlation between the expression of CaR and CAS mRNA levels.
Full figure and legend (16K)Expression levels within the individual glands
The variable expression within nodular glands is exemplified in Figure 1, which shows sections of three glands from patient 1 and corresponding controls hybridized in the same experiment and exposed to the same film. Six nodular areas (N1 to N6) and three internodular areas (I1 to I3) can be seen in these glands. N1 is an oxyphil nodule, whereas I1 is mainly composed of chief cells. The three nodules N2 to N4 and the internodular area I2 of gland 2 are all composed of chief cells. I3 of gland 3 consists of chief cells, whereas N6 is composed of oxyphil cells.
GAPDH mRNA is expressed at a higher level in the nodular than in the internodular areas of glands 1 and 3. No great differences in CaR mRNA can be seen between the nodular and internodular areas in any gland. The expression of CAS in this patient was extremely low. Only a faint expression can be identified in I2. VDR mRNA expression, on the other hand, is highly variable. All internodular areas of I1 to I3 have a low expression, whereas the nodular parts go from very low levels in N5, to low in N1, moderate in N3 and N4, and high in N2 and N6. The highest expression of PTH mRNA is seen in the nodular parts of gland 2. In gland 3, the level of PTH mRNA in the internodular area I3 is in between the two nodules N5 an N6.
Although the anticipated negative relationship between VDR and PTH mRNAs was observed in a few cases Figure 1b, the opposite was seen in other cases Figure 1c. No significant correlation between VDR and PTH mRNAs could be demonstrated.
The relationship between nodular and adjacent intermodular areas could be analyzed in nine glands. In 12 to 13 such pairs of nodular/internodular areas, the mRNA expression was higher in the nodular than in the internodular area of 10 out of 13 (GAPDH), 8 of 12 (CaR), 11 of 12 (VDR), and 11 of 13 (PTH) pairs. The results for VDR are shown in detail in Figure 4. Statistical analysis showed that the nodular areas had a significantly higher expression of all analyzed mRNAs (P < 0.05). The higher levels in nodular areas were not due to an increased density of cells in the nodules because the number of nuclei per unit area was the same in the nodular and internodular areas (results not shown).
Figure 4.
Vitamin D receptor mRNA expression in nodular parathyroids. A comparison between the nodular (
) and internodular (
) areas is shown.
The expression of CAS was generally low with no apparent differences between nodular and internodular areas, making identification of single nodules difficult.
One of the glands investigated was not hyperplastic due to uremia, but came from a young patient with hypophosphatemic rickets. This gland did not differ in any respect from those of the uremic patients.
DISCUSSION
This study investigated the expression of CaR, CAS, VDR, and PTH mRNA in hyperplastic parathyroid glands of patients with hypercalcemic secondary HPT. CaR, CAS, and VDR all take part in the intricate regulation of PTH synthesis and secretion and probably also in the proliferation of parathyroid cells in secondary HPT. Although we could only analyze a very few normal parathyroid biopsies, our results are in general agreement with previous studies demonstrating reduced levels of CaR12,14, CAS8, and VDR23,24,26,31,33, mRNA or protein in these glands in comparison with normal parathyroid glands. In addition, the present investigation reveals unexpected findings at the mRNA level in nodular glands that differ from previous results mainly at the protein level, but also in some cases at the mRNA level.
Reduced immunostaining for CaR protein has been demonstrated in primary as well as secondary HPT. Kifor et al reported that the staining intensity varied considerably between different glands, even in the same patient12. Only four glands from each of two patients with secondary HPT were analyzed, and no detailed description of the distribution in nodular glands was given. Gogusev et al reported that the expression of both CaR mRNA and protein was depressed in hyperplastic glands of secondary HPT14. With regard to CaR mRNA expression, the visual impression by light microscopy was that the decrease was, in some instances, more pronounced in the nodular areas than in surrounding areas of the four glands analyzed. However, this difference was not found using image analysis14. CaR protein staining was often lower in the nodular areas of five analyzed glands.
A recent short report by Akizawa et al analyzed the hyperplastic glands of six patients33. Although no numeric values were given in that report, it was stated that CaR protein expression was particularly depressed in the nodular lesions compared with diffuse lesions. A more profound reduction of CaR mRNA was demonstrated in large nodular hyperplastic glands than in small hyperplastic glands. The method used was Northern blot and Southern blot analysis after reverse transcriptase-polymerase chain reaction (RT-PCR). Thus, no information could be obtained about the relationship between the nodular and internodular areas.
Akizawa et al also reported that the expression of CaR protein was inversely correlated to cellular proliferation as indicated by proliferating cell nuclear antigen (PCNA)33. Similar findings were made in hyperplastic parathyroid glands of uremic rats fed with a phosphate-rich diet34, where CaR was decreased primarily in areas of active cell proliferation as indicated with PCNA. In line with that study, Yano et al recently reported a correlation between Ki67-positive cell numbers and CaR protein in secondary hyperplastic glands31.
To summarize previous studies on the expression of CaR protein, it is evident that CaR protein expression is reduced in secondary HPT compared with normal parathyroid glands. It also seems clear that CaR protein is more reduced in nodular areas than in diffusely hyperplastic areas of nodular glands. The reports concerning CaR mRNA on the other hand are ambiguous. In the present study, higher as well as lower expression of CaR mRNA was seen in nodules of nodular glands. Mean mRNA expression was slightly, but significantly higher in the nodular than in the internodular areas.
One possible explanation for the discrepancy between the present findings of expression at the mRNA level and previous histochemical findings at the protein level may be a different post-transcriptional handling of the receptor proteins in the nodular areas. A shorter half-life or else different processing of receptor protein in the nodules may be responsible. Whether the reduced expression of CaR protein is the driving force behind hyperplasia in the nodules or whether the reduced protein expression is only a secondary phenomenon has to be proven. Decreased expression of receptor molecules is a general and unspecific finding in rapidly proliferating cells.
The expression of CAS mRNA was markedly reduced in the hyperplastic glands when compared with the normal parathyroid glands. This finding is similar to that in sporadic adenomas where CAS mRNA expression was reduced to 25% of that in normal parathyroid biopsies from the same patient35.
The first report on reduced VDR in uremic HPT was by Korkor, who used a binding assay and could demonstrate significantly lower levels of VDR in hyperplastic parathyroid glands from patients with chronic renal failure than in glands from patients with transplanted kidneys, and especially in comparison with adenomas of patients with primary HPT23. Reduced levels of VDR protein as revealed by immunocytochemistry also have been reported by other investigators24,26. Fukuda et al found that VDR distribution was highly heterogeneous, with significantly lower levels in nodular than in diffusely hyperplastic tissue26. No detailed description on the relationship between different nodules and internodular areas was given. However, small nodules in diffusely hyperplastic glands were reported to be virtually negative with regard to VDR protein. Yano et al confirmed that VDR protein is decreased in parathyroid glands from secondary HPT, but did not demonstrate a significant difference between nodular and diffuse hyperplasia. In contrast, detailed analysis showed that nodular areas did not show a decreased expression of VDR compared with surrounding internodular areas31. Carling et al analyzed VDR mRNA in secondary HPT using RNase protection assay of whole glands and reported low levels compared with normal parathyroid and significantly lower levels in nodular than in diffusely hyperplastic tissue25. However, their results were all corrected for the level of GAPDH, which was used as an internal standard. In view of the variation in the expression of GAPDH mRNA demonstrated in the present investigation, caution should be taken when interpreting results after such corrections, since they may distort the results significantly.
Also, the expression of PTH mRNA was reduced in the hyperplastic glands when compared with the normal parathyroid biopsies. This deviates from the findings in experimentally induced secondary HPT of uremic animals in which the level of PTH mRNA is increased, mainly due to decreased degradation of mRNA36. However, the present results are in agreement with human studies, where the PTH content in sporadic adenomas has been shown to be significantly lower than in normal parathyroid glands even if no significant differences in mRNA levels could be detected37. The main reason for the high level of serum PTH in secondary HPT is probably the great increase in parathyroid cell mass and not an increased synthesis.
Vitamin D inhibits the transcription of the PTH gene via VDR19,20, and one would have expected a negative correlation between the expression of VDR and PTH mRNA. However, no such correlation was found. The lack of correlation could be explained by agonist action on the receptor, since treatment with vitamin D had been stopped well ahead of the surgery in most patients and serum levels of active vitamin D were probably low.
The present results show decreased levels of CAS, PTH and VDR mRNAs in the hyperplastic parathyroid tissue of secondary HPT compared with the levels in normal parathyroid glands. Furthermore, all the investigated mRNAs were heterogeneously expressed with slightly higher levels in nodular than in internodular areas. In spite of this finding, no correlation between the analyzed mRNAs could be demonstrated, except for the calcium-sensing receptors CaR and CAS. Thus, it seems that each nodule has its own profile of mRNA expression. In a certain nodule, VDR mRNA can be high, whereas CaR mRNA is low. In another nodule, it is the opposite. This is in good agreement with Yano et al, who found that the expression of CaR was independent of that of VDR31. The reason for the striking variation between nodules is unknown, but it cannot be attributed to humoral factors such as serum phosphate, calcium or active vitamin D. In view of the homogenous expression within each nodule a genetic reason must be anticipated. It is probable that the rapid proliferation caused by high serum phosphate/low serum calcium/low vitamin D increases the risk for genetic "mishappenings" that are then inherited by all cells in a certain nodule. The nature of these mutations is not known. No frequent large genetic events (gains or losses) have been found using comparative genomic hybridization of whole glands38 and allelic losses are infrequent in single nodules39. Although one report has indicated a role for the MEN1 region on chromosome 11q1340, this has not been verified as a common mechanism in other reports38,41. Our findings corroborate previous assumptions that each nodule in secondary HPT is of monoclonal origin, but that the clonal origin of each nodule is separate29.
The variation in the expression of receptor mRNAs of importance for the control of PTH secretion and parathyroid proliferation gives an explanation to the great variability in size of each parathyroid gland in the same patient. This finding also may explain the great variability in the clinical course of secondary HPT, where some patients can be adequately controlled by treatment with active vitamin D, while others are resistant to that treatment.
References
| 1. | Brown E. Four-parameter model of the sigmoidal relationship between parathyroid hormone release and extracellular calcium concentration in normal and abnormal parathyroid tissue. J Clin Endocrinol Metab 1983; 56: 572−581. | PubMed | ISI | ChemPort | |
| 2. | Brown E, Gamba G & Riccardi D et al. Cloning and characterization of an extracellular Ca2+-sensing receptor from bovine parathyroid. Nature 1993; 366: 575−580. | Article | PubMed | ISI | ChemPort | |
| 3. | Pollak M, Brown E & Chou Y et al. Mutations in the human Ca2+-sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Cell 1993; 75: 1297−1303. | Article | PubMed | ISI | ChemPort | |
| 4. | Hosokawa Y, Pollak M, Brown E & Arnold A. Mutational analysis of the extracellular Ca2+-sensing receptor gene in human parathyroid tumors. J Clin Endocrinol Metab 1995; 80: 3107−3110. | Article | PubMed | ISI | ChemPort | |
| 5. | Brown E, Pollak M & Chou Y et al. Cloning and functional characterization of extracellular Ca2+-sensing receptors from parathyroid and kidney. Bone 1995; 17: 7S−11S 10.1016/8756-3282(95)00199-N. | Article | PubMed | ChemPort | |
| 6. | Juhlin C, Johansson H & Holmdahl R et al. Monoclonal anti-parathyroid antibodies interfering with a Ca2+ sensor of human parathyroid cells. Biochem Biophys Res Commun 1987; 143: 570−574. | Article | PubMed | ISI | ChemPort | |
| 7. | Juhlin C, Holmdahl R & Johansson H et al. Monoclonal antibodies with exclusive reactivity against parathyroid cells and tubule cells of the kidney. Proc Natl Acad Sci USA 1987; 84: 2990−2994. | PubMed | ChemPort | |
| 8. | Juhlin C, Klareskog L & Nygren P et al. Hyperparathyroidism is associated with reduced expression of a parathyroid calcium receptor mechanism defined by monoclonal antiparathyroid antibodies. Endocrinology 1988; 122: 2999−3001. | PubMed | ISI | ChemPort | |
| 9. | Lundgren S, Hjälm G & Hellman P et al. A protein involved in calcium sensing of the human parathyroid and placental cytotrophoblast cells belongs to the LDL-receptor protein superfamily. Exp Cell Res 1994; 212: 344−350 10.1006/excr.1994.1153. | Article | PubMed | ISI | ChemPort | |
| 10. | Hjälm G, Murray E & Crumley G et al. Cloning and sequencing of human gp330, a Ca2+-binding receptor with potential intracellular signaling properties. Eur J Biochem 1996; 239: 132−137. | Article | PubMed | ISI | ChemPort | |
| 11. | Brown E, LeBoff M & Oetting M et al. Secretory control in normal and abnormal parathyroid tissue. Rec Prog Horm Res 1987; 43: 337−382. | PubMed | ISI | ChemPort | |
| 12. | Kifor O, Moore F, Jr & Wang P et al. Reduced immunostaining for the extracellular Ca2+-sensing receptor in primary and uremic secondary hyperparathyroidism. J Clin Endocrinol Metab 1996; 81: 1598−1606. | Article | PubMed | ISI | ChemPort | |
| 13. | Farnebo F, Enberg U & Grimelius L et al. Tumor-specific decreased expression of the calcium sensing receptor messenger ribonucleic acid in sporadic primary hyperparathyroidism. J Clin Endocrinol Metab 1997; 82: 3481−3486. | Article | PubMed | ISI | ChemPort | |
| 14. | Gogusev J, Duchambon P & Hory B et al. Depressed expression of calcium receptor in parathyroid gland tissue of patients with hyperparathyroidism. Kidney Int 1997; 51: 328−336. | PubMed | ISI | ChemPort | |
| 15. | Juhlin C, Rastad J & Klareskog L et al. Parathyroid histology and cytology with monoclonal antibodies recognizing a calcium sensor of parathyroid cells. Am J Pathol 1989; 135: 321−328. | PubMed | ISI | ChemPort | |
| 16. | Yamamoto M, Igarashi T & Muramatsu M et al. Hypocalcemia increases and hypercalcemia decreases the steady-state level of parathyroid hormone messenger RNA in the rat. J Clin Invest 1989; 83: 1053−1056. | PubMed | ISI | ChemPort | |
| 17. | Russel J, Lettieri D & Sherwood L. Direct regulation by calcium of cytoplasmic messenger ribonucleic acid coding for preproparathyroid hormone in isolated bovine parathyroid cells. J Clin Invest 1983; 72: 1851−1855. | PubMed | ISI | ChemPort | |
| 18. | Okazagi T, Ando K & Igarishi T et al. Conserved mechanism of negative regulation by extracellular calcium: Parathyroid hormone versus atrial natriuretic polypeptide gene. J Clin Invest 1992; 89: 1268−1273. | PubMed | ISI | ChemPort | |
| 19. | Silver J, Naveh-Many T & Mayer H et al. Regulation by vitamin D metabolites of parathyroid hormone gene transcription in vivo in the rat. J Clin Invest 1986; 78: 1296−1301. | PubMed | ISI | ChemPort | |
| 20. | Demay M, Kiernan M, DeLuca H & Kronenberg H. Sequences in the human parathyroid hormone gene that bind the 1,25-dihydroxyvitamin D3 receptor and mediate transcriptional repression in response to 1,25-dihydroxyvitamin D3. Proc Natl Acad Sci USA 1992; 89: 8097−8101. | PubMed | ChemPort | |
| 21. | Szabo A, Merke J & Beier E et al. 1,25(OH)2 vitamin D3 inhibits parathyroid cell proliferation in experimental uremia. Kidney Int 1989; 35: 1049−1056. | PubMed | ISI | ChemPort | |
| 22. | Ishimi Y, Russell J & Sherwood L. Regulation by calcium and 1,25-(OH)2D3 of cell proliferation and function of bovine parathyroid cells in culture. J Bone Miner Res 1990; 5: 755−760. | PubMed | ISI | ChemPort | |
| 23. | Korkor A. Reduced binding of [3H]1,25-dihydroxyvitamin D3 in the parathyroid glands of patients with renal failure. N Engl J Med 1987; 316: 1573−1577. | PubMed | ISI | ChemPort | |
| 24. | Fletcher S, Brownjohn A, Dunwell C & Harnden P. Immunocytochemical detection of a reduction in 1,25 dihydroxyvitamin D3 receptor in uraemic parathyroid tissue. Nephrol Dial Transplant 1997; 12: 93−96. | PubMed | ISI | ChemPort | |
| 25. | Carling T, Rastad J & Szabo E et al. Reduced parathyroid vitamin D receptor messenger ribonucleic acid levels in primary and secondary hyperparathyroidism. J Clin Endocrinol Metab 2000; 85: 2000−2003. | Article | PubMed | ISI | ChemPort | |
| 26. | Fukuda N, Tanaka H & Tominaga Y et al. Decreased 1,25-dihydroxyvitamin D3 receptor density is associated with a more severe form of parathyroid hyperplasia in chronic uremic patients. J Clin Invest 1993; 92: 1436−1443. | PubMed | ISI | ChemPort | |
| 27. | Slatopolsky EC, Weerts C & Thielan J et al. Marked suppression of secondary hyperparathyroidism by intravenous administration of 1,25-dihydroxycholecalciferol in uremic patients. J Clin Invest 1984; 74: 2136−2143. | PubMed | ISI | ChemPort | |
| 28. | Fukagawa M, Okazaki R & Takano K et al. Regression of parathyroid hyperplasia by calcitriol-pulse therapy in patients on long-term dialysis. N Engl J Med 1990; 323: 421−422. | PubMed | ISI | ChemPort | |
| 29. | Tominaga Y, Kohara S & Namii Y et al. Clonal analysis of nodular parathyroid hyperplasia in renal hyperparathyroidism. World J Surg 1996; 20: 744−752. | Article | PubMed | ISI | ChemPort | |
| 30. | Wallfelt C, Larsson R & Gylfe E et al. Secretory disturbance in hyperplastic parathyroid nodules of uremic hyperparathyroidism: Implications for parathyroid autotransplantation. World J Surg 1988; 12: 431−438. | Article | PubMed | ISI | ChemPort | |
| 31. | Yano S, Sugimoto T & Tsukamoto T et al. Association of decreased calcium-sensing receptor expression with proliferation of parathyroid cells in secondary parathyroidism. Kidney Int 2000; 58: 1980−1986. | Article | PubMed | ISI | ChemPort | |
| 32. | Dagerlind Å, Friberg K, Bean A & Hökfelt T. Sensitive mRNA detection using unfixed tissue: Combined radioactive and non-radioactive in situ hybridization histochemistry. Histochemistry 1992; 98: 39−49. | Article | PubMed | ISI | |
| 33. | Akizawa T, Ogata H & Koiwa F et al. The role of calcium-sensing receptor abnormality in the pathogenesis of secondary hyperparathyroidism. Nephrol Dial Transplant 1999; 14 Suppl 1: 66−67. | PubMed | ISI | |
| 34. | Brown A, Ritter C, Finch J & Slatopolsky E. Decreased calcium-sensing receptor expression in hyperplastic parathyroid glands of uremic rats: Role of dietary phosphate. Kidney Int 1999; 55: 1284−1292 10.1046/j.1523-1755.1999.00386.x. | Article | PubMed | ISI | ChemPort | |
| 35. | Farnebo F, Höög A & Sandelin K et al. Decreased expression of calcium-sensing receptor messenger ribonucleic acids in parathyroid adenomas. Surgery 1998; 124: 1094−1099 10.1067/msy.1998.91828. | Article | PubMed | ISI | ChemPort | |
| 36. | Yalcindag C, Silver J & Naveh-Many T. Mechanism of increased parathyroid hormone mRNA in experimental uremia: Roles of protein RNA binding and RNA degradation. J Am Soc Nephrol 1999; 10: 2562−2568. | PubMed | ISI | ChemPort | |
| 37. | Weber C, Russell J & Chryssochoos J et al. Parathyroid hormone content distinguishes true normal parathyroids from parathyroids of patients with primary hyperparathyroidsm. World J Surg 1996; 20: 1010−1015. | Article | PubMed | ISI | ChemPort | |
| 38. | FORSBERG L, VILLABLANCA A, V&AUML;LIM&AUML;KI S, et al: Homozygous inactivation of the MEN1 gene as a specific somatic event in a case of secondary hyperparathyroidism. Eur J Endocrinol (in press). |
| 39. | Chudek J, Ritz E & Kovacs G. Genetic abnormalities in parathyroid nodules of uremic patients. Clin Cancer Res 1998; 4: 211−214. | PubMed | ISI | ChemPort | |
| 40. | Falchetti A, Bale AE & Amorosi A et al. Progression of uremic hyperparathyroidism involves allelic loss on chromosome 11. J Clin Endocrinol Metab 1993; 76: 139−144. | Article | PubMed | ISI | ChemPort | |
| 41. | Farnebo F, Farnebo L-O, Nordenström J & Larsson C. Allelic loss on chromosome 11 is uncommon in parathyroid glands of patients with hypercalcaemic secondary hyperparathyroidism. Eur J Surg 1997; 163: 331−337. | PubMed | ISI | ChemPort | |
Acknowledgments
This work was supported by the Swedish Cancer Foundation, the Torsten and Ragnar Söderberg Foundations, the Swedish Medical Research Council, the Gustaf V Jubilee Foundation, the Fredrik and Ingrid Thuring Foundation, and the Cancer Society in Stockholm.
